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大鼠全层皮肤缺损创面愈合过程中巨噬细胞浸润及表型研究

Infiltration of macrophages and their phenotype in the healing process of full-thickness wound in rat

摘要:

目的 研究大鼠全层皮肤缺损创面愈合过程中巨噬细胞的浸润及表型变化. 方法 取30只健康SD大鼠,按随机数字表法分为损伤组24只、对照组6只.损伤组大鼠于脊柱两侧用自制环钻及手术剪制成2个直径为11 mm的全层皮肤缺损创面,致伤后即刻测量创面面积,每日碘伏消毒;对照组大鼠仅行麻醉脱毛处理.伤后1、3、7、13 d,分别取损伤组6只大鼠测量创面面积(计算创面愈合率)后处死,沿创缘切取创面组织达健康筋膜层,HE染色观察组织学表现,免疫组织化学染色观察组织中巨噬细胞表面标志物CD68表达,免疫荧光染色分别观察组织中CD68与诱导型一氧化氮合酶(iNOS)双阳性(Ⅰ型巨噬细胞)、CD68与精氨酸酶1(Arg-1)双阳性(Ⅱ型巨噬细胞)表达情况,双抗体夹心ELISA法检测创面组织中γ干扰素、TNF-α、IL-4、IL-13、IL-10和IL-12的水平并计算IL-10/IL-12比值.对照组大鼠在与损伤组相同部位切取直径为11 mm的全层正常皮肤组织,同前行组织学及细胞因子检测.对数据行单因素方差分析或LSD-t检验. 结果 损伤组大鼠伤后创面逐渐缩小,伤后各时相点创面愈合率总体比较差异有统计学意义(F=358.55,P<0.01).对照组大鼠皮肤组织形态未见异常.损伤组大鼠伤后1、3d,创面组织中炎性细胞明显浸润;伤后7、13d,可见明显血管腔结构、新生胶原.对照组大鼠正常组织和损伤组大鼠伤后1、3、7、13d创面组织每200倍视野下的CD68阳性细胞数分别为(2.7±1.5)、(31.8±3.5)、(40.8±4.7)、(20.8±2.8)、(3.2±2.4)个(F=180.55,P<0.01).损伤组大鼠伤后1、3、7 d CD68阳性细胞数明显高于对照组(t值分别为18.81、18.79、14.05,P值均小于0.01).对照组大鼠正常组织中未见CD68与iNOS双阳性或者CD68与Arg-1双阳性细胞.损伤组大鼠伤后1、3、7、13 d CD68与iNOS双阳性细胞百分比分别为(12.2±2.8)%、(16.5±2.9)%、(4.2±2.3)%、(0.7±0.8)%(F=72.50,P<0.01),CD68与Arg-1双阳性细胞百分比分别为0、(8.2±1.9)%、(21.5±3.4)%、(4.7±2.0)%(F=120.93,P<0.01).损伤组大鼠伤后3 d CD68与iNOS双阳性细胞百分比显著高于组内其他时相点(t值分别为2.65、8.17、12.95,P值均小于0.05),伤后7 d CD68与Arg-1双阳性细胞百分比显著高于组内其他时相点(t值分别为15.27、8.25、10.38,P值均小于0.01).CD68与iNOS双阳性细胞百分比于伤后1、3d显著高于CD68与Arg-1双阳性细胞百分比(t值分别为10.71、5.88,P值均小于0.01),伤后7、13d则显著低于CD68与Arg-1双阳性细胞百分比(t值分别为10.24、4.60,P值均小于0.01).对照组大鼠正常组织及损伤组大鼠伤后各时相点创面组织中γ干扰素、TNF-α、IL-4、IL-13水平及IL-10/IL-12比值总体比较,差异均有统计学意义(F值为14.08 ~631.03,P值均小于0.01).与对照组比较,损伤组大鼠伤后各时相点创面组织中γ干扰素、TNF-α、IL-4和IL-13水平均显著增高(t值为4.58~ 9.17,P值均小于0.05),伤后1、3、7 d IL-10/IL-12比值明显增高(t值分别为27.70、30.51、9.49,P值均小于0.05).损伤组大鼠伤后1dγ干扰素水平[(61±5) pg/mL]及伤后3 d IL-10/IL-12比值(1.647 ±0.098),显著高于对照组及损伤组其余时相点[γ干扰素水平依次为(32±4)、(54±6)、(46±7)、(47 ±4) pg/mL,IL-10/IL-12比值依次为0.328±0.045、0.960±0.034、0.530±0.028、0.289±0.040,t值分别为3.19 ~ 8.20、16.59 ~31.84,P值均小于0.05]. 结论 大鼠全层皮肤缺损创面愈合过程中巨噬细胞浸润明显增加并呈现不同表型,其中Ⅰ型巨噬细胞出现在炎症期,而增殖期以Ⅱ型巨噬细胞为主.

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abstracts:

Objective To study the infiltration of macrophages and their phenotype in the healing process of full-thickness wound in rat.Methods Thirty healthy SD rats were divided into control group (n =6) and injury group (n =24) according to the random number table.Two round full-thickness skin defects (11 mm diameter) were created on both sides of dorsal spine of rats in injury group with surgical scissors and homemade trephine.After injury,wound area was measured immediately.The wounds were disinfected with iodophor every day.Rats in control group received anesthesia and hair removal only.On post injury day (PID) 1,3,7,and 13,respectively,6 rats of injury group were sacrificed after the measurement of wound area (wound healing rate was calculated).Wound samples were obtained by excision down to healthy fascia along wound edge.Histological study was done with HE staining.The expression of CD68 (the surface marker of macrophage) in the wound tissue was observed with immunohistochemical staining.The double positive expressions of induced nitric oxide synthase (iNOS) plus CD68 (type Ⅰ macrophage) and arginase 1 (Arg-1)plus CD68 (type Ⅱ macrophage)were observed with immunofluorescence staining.The levels of interferon-γ (IFN-γ),TNF-o,IL-4,IL-13,IL-10,and IL-12 in wound tissue were assayed by double-antibody sandwich ELISA,and the ratio of IL-10/IL-12 was calculated.Full-thickness skin tissues (11 mm diameter) in rats of control group were excised at the same site as rats in injury group,and the histological observation and cytokines assay were performed as well.Data were processed with one-way analysis of variance or LSD-t test.Results Wound area of rats in injury group was gradually reduced after injury,and the overall difference of the wound healing rate on each PID was statistically significant (F =358.55,P <0.01).No abnormal appearance of skin tissue was observed in rats of control group.In injury group,inflammatory cell infiltration was obvious in wound tissue on PID 1 and 3; vascular structure and fresh collagen were observed in wound tissue on PID 7 and 13.Numbers of CD68 positive cells in skin tissue of rats in control group and wound tissue of rats in injury group on PID 1,3,7,and 13 were respectively (2.7±1.5),(31.8 ±3.5),(40.8 ±4.7),(20.8 ±2.8),(3.2 ±2.4) per 200 times visual field (F =180.55,P <0.01).Compared with that in control group,the number of CD68 positive cells of rats in injury group was increased on PID 1,3,and 7 (with t values respectively 18.81,18.79,14.05,P values below 0.01).No double positive expression of iNOS plus CD68 or Arg-1 plus CD68 was observed in normal tissue of rats in control group.In injury group,proportions of iNOS plus CD68 double positive cells on PID 1,3,7,and 13 were respectively (12.2±2.8)%,(16.5 ±2.9)%,(4.2 ±2.3)%,(0.7 ±0.8)% (F =72.50,P <0.01); proportions of Arg-1 plus CD68 double positive cells on PID 1,3,7,and 13 were respectively 0,(8.2±1.9)%,(21.5±3.4)%,(4.7±2.0)% (F =120.93,P <0.01).In injury group,proportion of iNOS plus CD68 double positive cells on PID 3 was significantly higher than that on other PID (with t values respectively 2.65,8.17,12.95,P values below 0.05) ; proportion of Arg1 plus CD68 double positive cells on PID 7 was higher than that on other PID (with t values respectively 15.27,8.25,10.38,P values below 0.01).Compared with that of Arg-1 plus CD68 double positive cells,proportion of iNOS plus CD68 double positive cells was higher on PID 1 and 3 (with t values respectively 10.71 and 5.88,P values below 0.01) and lower on PID 7 and 13 (with t values respectively 10.24 and 4.60,P values below 0.01).The overall differences of IFN-γ,TNF-α,IL-4,IL-13,and IL-10/IL-12 ratio in skin tissue of rats in control group and wound tissue of rats in injury group on every PID were statistically significant (with F values from 14.08 to 631.03,P values below 0.01).Compared with those in control group,levels of IFN-γ,TNF-α,IL-4,and IL-13 in wound tissue of rats in injury group were significantly higher on every PID (with t values from 4.58 to 9.17,P values below 0.05),while IL-10/IL-12 ratio was significantly higher on PID 1,3,and 7 (with t values respectively 27.70,30.51,9.49,P values below 0.05).In injury group,IFN-γ level on PID 1 [(61 ± 5) pg/mL] and IL-10/IL-12 ratio on PID 3 (1.647 ± 0.098)were significantly higher than those of control group and those on other PID in injury group [with IFN-γ level respectively (32 ±4),(54 ±6),(46 ±7),(47 ±4) pg/mL and IL-10/IL-12 ratio respectively0.328 ± 0.045,0.960 ±0.034,0.530 ±0.028,0.289 ±0.040,with t values respectively from 3.19 to 8.20 and from 16.59 to 31.84,P values below 0.05].Conclusions Macrophage infiltration increases in the healing process of full-thickness wound in rat with different phenotypes,among which type Ⅰ macrophage appears in the inflammatory stage,and type Ⅱ macrophage predominates in the proliferative stage.

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作者: 牛轶雯 [1] 缪明远 [2] 曹晓赞 [1] 宋菲 [1] 嵇晓芸 [1] 董叫云 [1] 陆树良 [1]
期刊: 《中华烧伤杂志》2014年30卷2期 109-115页 MEDLINEISTICPKUCSCD
栏目名称: 创面修复的基础与临床研究
DOI: 10.3760/cma.j.issn.1009-2587.2014.02.004
发布时间: 2014-05-13
基金项目:
国家自然科学基金面上项目 上海市卫生局青年科研项目
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